[Reconstitution of rabbit brain GABA binding protein (receptor) on Xenopus oocytes]

Gurdon's Xenopus laevis oocyte translation system has been extensively employed to investigate the molecular biology of gamma-aminobutyric acid (GABA) receptors in the brains of embryonic chick, rat, etc. As GABA and its receptor may play a role in the pathogenesis of hepatic encephalopathy and a rabbit model of fulminant hepatitis has been established, we reconstituted the rabbit model of fulminant hepatitis has been established, we reconstituted the rabbit brain GABA binding protein (receptor) on Xenopus laevis oocytes to offer a base for further study of the pathological effects of GABA receptor at molecular level. In this report, total RNA was extracted from rabbit brain by guanidine hydrochloride method. The total RNA was then subjected to oligo-(dT)-cellulose affinity chromatography to isolate mRNA. All the RNA samples achieved a high purity with an average OD260/OD280 ratio of about 2.10. The clear bands of 18 S and 28S rRNA in electrophoresis implied that the RNA was not degraded and was therefore available for expression. After two days of incubation of the oocytes injected with mRNA, radioreceptor assay indicated that saturable and specifically displaceable GABA binding sites were implanted onto the oocyte membrane. This result led us to the conclusion that rabbit brain GABA binding protein (receptor) can be reconstituted by injecting exogenous mRNA inclusive of those encoding the receptor into Xenopus oocytes. However, the functional activity of the reconstituted receptor as a GABA gated chloride ion channel needs to be further characterized by patch clamping.